Expression of eukaryotic peptides in plant plastids

ABSTRACT

Constructs and methods are provided for expressing peptides derived from eukaryotic organisms in plant plastids. Constructs have a promoter functional in a plant plastid, a DNA sequence encoding a peptide derived from an eukaryotic organism and a transcription termination region. Other elements include a selectable marker for selection of plant cells comprising a plastid expressing the marker and DNA regions of homology to the genome of the plastid and optionally a ribosome binding site joined to the promoter. By methods using such constructs high levels of eukaryotic peptides, such as mammalian proteins, are produced in a plant cell by growing plant cells under conditions whereby the DNA encoding sequences are expressed to produce eukaryotic peptide in said plastid.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/103,516, filed Mar. 20, 2002, now U.S. Pat. No. 6,812,379; which is acontinuation of U.S. patent application Ser. No. 09/316,847 filed May21, 1999, now abandoned; which is a continuation-in-part of U.S. patentapplication Ser. No. 09/113,244, filed Jul. 10, 1998, now U.S. Pat. No.6,512,162.

INTRODUCTION

1. Technical Field

This invention relates to the application of genetic engineeringtechniques to plants. Specifically, the invention relates tocompositions and methods for enhancing expression of proteins in plantplastids.

2. Background

The plastids of higher plants are an attractive target for geneticengineering. Plant plastids (chloroplasts, amyloplasts, elaioplasts,etioplasts, chromoplasts, etc.) are the major biosynthetic centers that,in addition to photosynthesis, are responsible for production ofindustrially important compounds such as amino acids, complexcarbohydrates, fatty acids, and pigments. Plastids are derived from acommon precursor known as a proplastid and thus the plastids present ina given plant species all have the same genetic content. Plant cellscontain 500-10,000 copies of a small 120-160 kilobase circular genome,each molecule of which has a large (approximately 25 kb) invertedrepeat. Thus, it is possible to engineer plant cells to contain up to20,000 copies of a particular gene of interest which potentially canresult in very high levels of foreign gene expression. In addition,plastids of most plants are maternally inherited. Consequently, unlikeheterologous genes expressed in the nucleus, heterologous genesexpressed in plastids are not pollen disseminated, therefore, a traitintroduced into a plant plastid will not be transmitted to wild-typerelatives.

There remains a need for improved regulatory elements for expression ofgenes in a plant plastid. To date, the expression signals used routinelyfor plastid transgene expression derive from endogenous plastid genes.The plastid expression signals are typically derived from promoterregions of highly expressed plastid genes such as the promoter regionsfrom the 16S ribosomal RNA operon (Prrn), psbA gene (PpsbA) or the rbcLgene (PrbcL). The psbA and rbcL genes are highly transcribed, but theirtranslation is controlled by tissue-specific and light-regulated factorswhich limits their usefulness. In the case of Prrn, a synthetic ribosomebinding site (RBS) patterned after the plastid rbcL gene leader has beentypically used to direct translation. However, this Prrn/RBS istranslated inefficiently due to poor ribosome binding.

Plastids of higher plants present an attractive target for geneticengineering. As mentioned above, plastids of higher plants arematernally inherited. This offers an advantage for genetic engineeringof plants for tolerance or resistance to natural or chemical conditions,such as herbicide tolerance, as these traits will not be transmitted towild-type relatives. In addition, the high level of foreign geneexpression is attractive for engineered traits such as the production ofpharmaceutically important proteins.

Expression of nucleic acid sequences encoding for enzymes providing forherbicide tolerance as well as pharmaceutical proteins from plantplastid genome offers an attractive alternative to expression from theplant nuclear genome.

Relevant Literature

McBride et al. U.S. Pat. No. 5,576,198 and McBride et al. (1994) ProcNatl Acad Sci 91:7301-7305 reports the plastid expression system basedon a two component system utilizing a nuclearly encoded T7 polymerasetargeted to the plastid which activates a transgene controlled by the T7bacteriophage gene 10 promoter. Svab et al. (1990) Proc Natl Acad Sci87:8526-8530 reports the standard chloroplast transformation methods.Svab, et al. (1993) Proc Natl Acad Sci 90:913-917 reports the use of theaadA gene for use in selection of transplastomic plants on spectinomycinand streptomycin, as well as integration sequences. Zoubenko et al.(1994) Nuc Acid Res 22:3819-3824 reports the construct of vectors foruse in plastid transformation.

Barry, et al., U.S. Pat. No. 5,627,061 describes the cloning of EPSPSnucleotide sequences from several sources including Agrobacterium strainCP4 and methods for producing glyphosate tolerant plants. Kishore andShah, Ann. Rev. Biochem. (1988) 57:627-663 reports the modification ofDNA sequences for the enzyme 5-enolpyruvylshikimate-3-phosphate synthase(hereinafter referred to as EPSP synthase or EPSPS) for the enhancementof glyphosate tolerance. Stalker et al., U.S. Pat. No. 4,810,648describes the cloning and use of a nucleic acid sequence encoding forthe bromoxynil degrading gene, nitrilase.

SUMMARY OF THE INVENTION

The present invention provides nucleic acid sequences useful inenhancing expression of a wide variety of genes, both eukaryotic andprokaryotic, in plant plastids. Furthermore, plastid expressionconstructs are provided which are useful for genetic engineering ofplant cells and which provide for enhanced expression of the EPSPsynthase proteins or the hGH protein in plant cell plastids. Thetransformed plastids should be metabolically active plastids, and arepreferably maintained at a high copy number in the plant tissue ofinterest, most preferably the chloroplasts found in green plant tissues,such as leaves or cotyledons. The plastid expression constructs for usein this invention generally include a plastid promoter region capable ofproviding for enhanced expression of a DNA sequence, a DNA sequenceencoding an EPSPS protein or human growth hormone (hGH), and atranscription termination region capable of terminating transcription ina plant plastid.

The plastid promoter region of the present invention is preferablylinked to a ribosome binding site which provides for enhancedtranslation of mRNA transcripts in a plant plastid.

The plastid expression construct of this invention is preferably linkedto a construct having a DNA sequence encoding a selectable marker whichcan be expressed in a plant plastid. Expression of the selectable markerallows the identification of plant cells comprising a plastid expressingthe marker.

In a preferred embodiment, vectors for transfer of the construct into aplant cell include means for inserting the expression and selectionconstructs into the plastid genome. The vectors preferably compriseregions of homology to the target plastid genome which flank theconstructs.

The constructs of the present invention preferably comprise a promotersequence linked to a ribosome binding site capable of enhancing thetranslation of mRNA transcripts in the plant plastid. The ribosomebinding site is preferably derived from the T7 bacteriophage gene 10leader sequence.

Of particular interest in the present invention is the high level ofexpression of nucleic acid sequences in plant plastids. Of particularinterest is the high level expression of nucleic acid sequences encodingfor enzymes involved in herbicide tolerance and encoding forpharmaceutical proteins.

The constructs of the present invention preferably comprise a DNAsequence encoding 5-Enolpyruvylshikimate-3-phosphate synthase (U.S. Pat.No. 5,633,435, the entirety of which is incorporated herein byreference), nitrilase, phytoene desaturase, aprotinin or a DNA sequenceencoding human growth hormone (U.S. Pat. No. 5,424,199, the entirety ofwhich is incorporated herein by reference).

Plant cell plastids containing the constructs are also contemplated inthe invention, as are plants, plant seeds, plant cells or progenythereof containing plastids comprising the construct.

The present invention also includes methods for enhanced expression ofDNA sequences in plant plastids.

The invention also includes a method for the enhanced expression of anenzyme encoding hGH in plastids of the plant cell.

The present invention further includes methods for obtaining a proteinexpressed from a plant cell, including a plastid, having anon-methionine N-terminus. In addition, plant cells and plastids whichinclude non-methionine N-terminus proteins are contemplated.

Thus, the present invention relates to a chimeric gene containing acoding sequence of a pharmaceutical protein, a plant plastid expressionvector containing a promoter operably linked to a T7 BacteriophagePolymerase gene 10 ribosome binding site capable of enhanced expressionin a plant plastid operably linked to a herbicide tolerance orpharmaceutical coding gene, a plant transformation vector havinginserted therein a herbicide tolerance or pharmaceutical coding geneexpressed from a plastid promoter linked to a T7 BacteriophagePolymerase gene 10 ribosome binding site, plant cells transformed usingsuch vectors and plants regenerated therefrom which exhibit asubstantial degree of expression of nucleic acid sequences and proteinsand methods for producing such plants and such plants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequence of the G10L ribosome binding site.

FIG. 2 shows a schematic of the plastid expression vector pMON38773.

FIG. 3 shows a schematic of the plastid expression construct pMON30123.

FIG. 4 shows a schematic of the plastid expression construct pMON30130.

FIG. 5 shows a schematic of the plastid expression construct pMON38773.

FIGS. 6A-6B show a schematic of the expression construct pWRG4747.

FIGS. 7A-7B show a schematic of the expression construct pWRG4838.

FIG. 8 shows a schematic of the expression construct pMON38755.

FIG. 9 shows a schematic of the expression construct pMON38794.

FIG. 10 provides a nucleic acid sequence encoding for aprotinin.

FIG. 11 provides the results of RP-HPLC analysis for characterization ofhGH protein expressed in the plastid. Peak I (tallest peak) indicatesthe expected retention time for properly folded, native 22 kDa GP2000.

FIG. 12 provides an electrospray ionization mass spectrometry (MS)analysis using a Micromass Q-T of electrospray time-of-flight massspectrometer. In particular, a series of ions corresponding to thespecie(s) present in the sample with varying numbers of protons attachedis provided. The axes of the spectrum are intensity versusmass-to-charge ratio of the specie(s) present.

FIG. 13 provides a graphic representation of the bioactivity of hGHexpressed from a plant plastid. The samples represented on the graph arebovine prolactin (bPL), hGH expressed from E. coli (Ala-hGH), and a nulltransgenic spiked with bovine prolactin (SPFF Null Spike) as positivecontrols, a null transgenic (SPFF Null) as a negative control, andtransgenic samples from a sepharose column (SPFF Sample, SPFF Sample)and a transgenic sample eluted from the sepharose column at pH3.5 (SPFFpH3.5 Eln).

FIGS. 14A-14B provide a Western Blot analysis of aprotinin expression inplastids.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the subject invention, plastid expression constructsare provided which generally comprise a promoter functional in a plantplastid, a ribosome binding site derived from the T7 BacteriophagePolymerase gene 10 leader, a DNA sequence encoding for a gene ofinterest, and a transcription termination region capable of terminatingtranscription in a plant plastid. These elements are provided asoperably joined components in the 5′ to 3′ direction of transcription.

Furthermore, the constructs of the present invention may also include anucleic acid sequence encoding a peptide capable of targeting said DNAsequence encoding a protein to the thylakoid lumen within thechioroplast.

Of particular interest in the present invention are methods for theproduction of proteins in a host plant cell plastid having anon-methionine N-terminus. Such methods generally involve the use offusion proteins having an N-terminus sequence which is recognized by anendogenous protease. In particular, a DNA sequence encoding acleavableubiquitin peptide is fused to a DNA sequence encoding a protein ofinterest. After expression of the fusion protein in the plastid, anendogenous protease acts on the fusion to cleave off the ubiquitinportion of the protein.

Also of interest in the present invention is the use of the plastidexpression constructs to direct the high level transcription andtranslation (expression) of nucleic acid sequences. Such plastidexpression constructs find use in directing the high level expression ofDNA sequences encoding for enzymes involved in herbicide tolerance orencoding for the production of pharmaceutical proteins.

Of more particular interest in the present invention is the use of theplastid expression constructs to direct the high level translation oftranscribed messenger RNA.

DNA sequence and biochemical data reveal a similarity of the plastidorganelle's transcriptional and translational machineries and initiationsignals to those found in prokaryotic systems. In fact, plastid derivedpromoter sequences have been reported to direct expression of reportergenes in prokaryotic cells. In addition, plastid genes are oftenorganized into polycistronic operons as they are in prokaryotes.

Despite the apparent similarities between plastids and prokaryotes,there exist fundamental differences in the methods used to control geneexpression in plastids and prokaryotes. As opposed to thetranscriptional control mechanisms typically observed in prokaryotes,plastid gene expression is controlled predominantly at the level oftranslation and mRNA stability by trans-acting nuclear encoded proteins.

Translation is a multi-stage process which first involves the binding ofmessenger RNA (mRNA) to ribosomes. Beginning at the translation startcodon, the mRNA codons are read sequentially as the ribosomes move alongthe mRNA molecule. The specified amino acids are then sequentially addedto the growing polypeptide chain to yield the protein or polypeptideencoded in the mRNA.

As mentioned, the first step in the translation process is the bindingof the mRNA molecule to the ribosome. The nature of this interaction(i.e. binding) has been only partially elucidated. Analysis ofRNase-resistant oligonucleotides isolated from bacterial translationinitiation complexes indicate that a RNA fragment approximately 30 to 40nucleotides in length comprises the initial ribosome binding site (RBS).Thus, a RBS is hereinafter understood to comprise a sequence of mRNAsurrounding the translation start codon which is responsible for thebinding of the ribosome and for initiation of translation.

Recently, ribosome binding sites have been identified which are capableof directing translation in a prokaryotes. For examples a ribosomebinding site derived from the 17 bacteriophage gene 10 leader, G10L(U.S. Pat. No. 5,232,840, the entirety of which is incorporated hereinby reference), has been identified which enhances expression of nucleicacid sequences in prokaryotes.

Herbicides such as N-phosphonomethylglycine, halogenatedhydroxybenzonitriles, and norflurazon have been the subject of a largeamount of investigation.

N-phosphonomethylglycine, commonly referred to as glyphosate, inhibitsthe shikimic acid pathway which leads to the biosynthesis of aromaticcompounds including amino acids, plant hormones and vitamins.Specifically, glyphosate curbs the conversion of phosphoenolpyruvic acid(PEP) and 3-phosphoshikimic acid to 5-enolpyruvyl-3-phosphoshikimic acidby inhibiting the enzyme 5-enolpyruvylshikimate-3-phosphate synthase(hereinafter referred to as EPSP synthase or EPSPS).

Glyphosate tolerant plants have been produced by transformation ofvarious EPSP synthase genes into the nuclear genome of a plant. A genefor EPSP synthase has been cloned from Agrobacterium tumefaciens spstrain CP4 (U.S. Pat. No. 5,633,435) and confers a high level ofglyphosate tolerance in plants. Furthermore, high levels of glyphosatetolerance has been achieved in a number of crop plants by fusing EPSPSto a chloroplast transit peptide (CTP) for targeted expression inplastids. In addition, variants of the wild-type EPSPS enzyme have beenisolated which are glyphosate tolerant as a result of alterations in theEPSPS amino acid coding sequence (Kishore and Shah, Ann. Rev. Biochem.(1988) 57:627-663; Shulze et al., Arch. Microbiol. (1984) 137:121-123;Kishore et al., Fed. Proc. (1986) 45:1506). These variants typicallyhave a higher K_(i) for glyphosate than the wild-type EPSPS enzyme whichconfers the glyphosate tolerant phenotype, but these variants are alsocharacterized by a high K_(m) for PEP which makes the enzyme kineticallyless efficient (Kishore and Shah, Ann. Rev. Biochem. (1988) 57:627-663;Sost et al., FEBS Lett. (1984) 173: 238-241; Shulze et al., Arch.Microbiol. (1984) 137:121-123; Kishore et al., Fed. Proc. (1986)45:1506; Sost and Amrhein, Arch. Biochem. Biophys. (1990) 282: 433-436).

In addition to engineering plants for glyphosate tolerance, plants havealso been engineered to tolerate other classes of herbicides such ashalogenated hydroxybenzonitriles, and norflurazon using nucleic acidsequences expressed in the nucleus.

Halogenated hydroxybenzonitriles, such as Bromoxynil, are suggested toact herbicidally by inhibiting the quinone-binding protein complex ofphotosystem II, inhibiting electron transfer (Van Rensen (1982) Physiol.Plant 54:515-520,and Sanders and Pallett (1986) Pestic. Biochem.Physiol. 26:116-122). Herbicides such as norflurazon inhibit theproduction of carotenoids.

Plants which are resistant to Bromoxynil have been produced byexpressing DNA sequences encoding for enzymes capable of detoxifyingBromoxynil (nitrilases) in the plant cell nucleus. DNA sequencesencoding for such nitrilases have been cloned from bacteria such asKlebsiella pneumoniae and used to construct vectors to direct theexpression of the DNA sequence in plant cell nucleus (U.S. Pat. No.4,810,648, the entirety of which is incorporated herein by reference).

Plants which are resistant to Norflurazon have been engineered byexpressing nucleic acid sequences which encode for enzymes in thecarotenoid biosynthetic pathway in plant cell nuclei. For example,expressing a phytoene desaturase from Erwinia uredovora providestolerance to norflurazon.

While plants transformed to express nucleic acid sequences encoding forsuch enzymes from the nuclear genome have found utility in engineeringherbicide tolerant plants, it would be increasingly beneficial to obtainherbicide tolerant plants via plastidial expression.

In the examples provided herein, DNA sequences encoding for enzymesinvolved in herbicide tolerance are used in constructs to direct theexpression of the sequences from the plant plastid. DNA sequencesencoding for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS),bromoxynil nitrilase (Bxn), phytoene desaturase (crtI (Misawa et al,(1993) Plant Journal 4:833-840, and (1994) Plant Jour 6:481-489), andacetohydroxyacid synthase (AHAS (Sathasiivan et al. (1990) Nucl. AcidsRes. 18:2188-2193)) are used in the expression constructs of the presentinvention to direct the expression of said herbicide tolerancenucleotide sequences from the plant plastid.

Transplastomic tobacco plants are identified which are homoplasmic forthe DNA sequences encoding the herbicide tolerance genes. Homoplasmicplants demonstrate a high level of protein expression from the plastid.Furthermore, homoplasmic plants demonstrate a high level of tolerancefor the respective herbicide. For example, as described in more detailin the example below, plants transformed to express EPSPS from theplastid demonstrate a high level of tolerance for the herbicideglyphosate. In addition, homoplasmic tobacco lines expressing nitrilaseor phytoene desaturase demonstrate high levels of tolerance for theherbicides bromoxynil and norflurazon, respectively.

An artisan skilled in the art to which the present invention pertainswill recognize that additional sequences may be employed to in theplastid expression constructs of the instant invention to produceherbicide tolerant plants. Other nucleic acid sequence which may finduse in the plastid expression constructs herbicide tolerant plantsinclude the bar gene for tolerance to glufosinate (DeBlock, et al.(1987) EMBO J. 6:2513-2519).

Furthermore, additional glyphosate tolerance genes may be employed inthe constructs of the present invention. Additional glyphosate tolerantEPSPS genes are described in U.S. Pat. No. 5,627,061, Padgette et al.(1996) Herbicide Resistant Crops, Lewis Publishers, 53-85, and inPenaloza-Vazquez, et al. (1995) Plant Cell Reports 14:482-487, theentireties of which are incorporated herein by reference.

It should be noted that the herbicide tolerance constructs of thepresent invention may also include sequences encoding genes involved inother stress tolerance genes, for example insect or diseaseresistance/tolerance genes. As described in more detail in the examplesthat follow, plastid expression constructs are used to regenerate plantswhich are resistant to the herbicide Buctril, and which also express theBacillus thuringensis crylAc protein.

In addition, the plastid expression constructs also find use indirecting the production of human biological proteins (pharmaceuticalproteins) from the plant plastid. As set forth in detail in theexamples, constructs are provided for expression of aprotinin and humangrowth hormone in the plant plastid. Other sequences which may find usein the expression constructs of the present invention for the productionof human biologics include sequences encoding for insulin or insulinprecursors. However, the skilled artisan will recognize that manynucleotide sequences encoding for human biologics may be employed in theconstructs of the present invention to direct their expression from aplant plastid such as those described in Goodman and Gelman (1990)Pharmacological Basis of Therapeutics, Pergaman Press, 8^(th) Edition,Sections 14 and 15. As, it is contemplated that any protein for whichthe nucleotide sequence has been identified can be used in theconstructs of the present invention.

The present invention also provides methods for producing apharmaceutical protein with a non-methionine N-terminus in a plantplastid. In general, the methods comprise expressing a fusion proteinincluding a ubiquitin gene fused to a protein of interest in a plastid.The ubiquitin gene is obtained from a natural source and cloned into anappropriate vector, as described in WO 88/02406, supra, the disclosureof which is incorporated herein by reference, or it is synthesizedchemically, using, e.g., the method described by Ecker et al., J. Biol.Chem., 262:3524-3527 (1987) and Ecker et al., J. Biol. Chem., 262:14213-14221 (1987), the disclosures of which are incorporated byreference. The ubiquitin fusion proteins are recognized by ubiquitinprotease, contrary to previous reports (Vierstra (1996) Plant Mol. Biol.32:275-302), which cleaves immediately downstream of the carboxyterminal glycine residue of ubiquitin. This property has allowedproduction of recombinant proteins containing N-terminal residues otherthan methionine (Baker (1996) Current Opin. Biotech 7:541-546).

Additional methods for the production of pharmaceutical proteins with anon-methionine N-terminus in a plant plastid are also provided. Asdescribed more fully in the Examples below, constructs are prepared todirect the production of a methionine-hGH (M-hGH) in a plant cellplastid. The constructs generally comprise a transcriptional initiationregion and a DNA sequence encoding hGH: Surprisingly, N-terminal aminoacid sequencing of the extracted hGH produced in transplastomic plantsreveals that the N-terminal methionine is cleaved from the mature hGHprotein, producing hGH with an alanine N-terminus (A-hGH). This resultindicates the interaction of the expressed hGH with a methionine aminopeptidase (MAP) in the plant cell. While it is anticipated that anyamino acid may follow in the N-terminal methionine, the second aminoacid is preferably selected from the group consisting of alanine,cysteine, glycine, proline, serine, threonine, and valine.

As described in more detail below, nucleic acid sequences encoding forthe human growth hormone (hGH) are employed in plastid expressionconstructs of the present invention. Further, transplastomic tobaccoplants containing such constructs demonstrate a high level of expressionof hbH. In addition, the hGH protein expressed from the plant plastidexhibits characteristics of proper processing as well as proper proteinfolding.

Human growth hormone (hGH) participates in much of the regulation ofnormal human growth and development. This 22,000 dalton pituitaryhormone exhibits a multitude of biological effects including lineargrowth (somatogenesis), lactation, activation of macrophages,insulin-like and diabetogenic effects among others (Chawla, Ann. Rev.Med. (1983) 34:519; Edwards, et al., Science (1988) 239:769; Thorner etal., J. Clin. Invest. (1988) 81:745). hGH is a member of a family ofhomologous hormones that include placental lactogens, prolactins, andother genetic and species variants or growth hormone (Nicoll, et al.,Endocrine Reviews (1986) 7:169). hGH is unusual among these in that itexhibits broad species specificity and binds to either the clonedsomatogenic (Leung, et al., Nature (1987) 33:537) or prolactin receptor(Boutin, et al., Cell (1988) 53:69). The primary use of hGH is in thetreatment of hypopituitary dwarfism in children. Additional indicationsare in treatment of Turner syndrome, chronic renal failure, HIV wastingsyndrome and the treatment of the elderly and critically ill (Tritos, etal. (1998) Am. J. Med. 105:44-57).

As produced in the pituitary gland, hGH enters the secretory system,coincident with removal of its signal peptide and formation of twodisulfide bonds (Chawla, et al. (1983) supra). In the pituitary gland,removal of the signal peptide from hGH (also referred to as humansomatotropin or hST) during secretion leaves phenylalanine as theN-terminal amino acid (Chawla, et al. (1983) Annu. Rev. Med.34:519-547). As normal translation in plastids initiates at methionine,a ubiquitin-hGH fusion was designed to yield a phenylalanine N-terminus(F-hGH) in the final hGH product.

Surprisingly, although ubiquitin protease was previously reported to notbe present in chloroplasts (Vierstra (1996) Plant Mol. Biol.32:275-302), the ubiquitin-hGH fusion was processed during synthesis,accumulation or purification from the plants to produce a phenylalanineN-terminus hGH product (F-hGH). The control construct carrying thefull-length cDNA encoded methionine and alanine as the first amino acidsof hGH.

As described in the Examples below, constructs comprising nucleic acidsequences encoding aprotinin (also known as bovine pancreatic trypsininhibitor, BPTI) were employed in plastid expression constructs of thepresent invention. Aprotinin is a basic protein present in severalbovine organs and tissues, such as the lymph nodes, pancreas, lungs,parotid gland, spleen and liver. Aprotinin is known to inhibit variousserine proteases, including trypsin, chymotrypsin, plasmin andkallikrein, and is used therapeutically in the treatment of acutepancreatitis, various stages of shock syndrome, hyperfibrinolytichemorrhage and myocardial infarction. In addition, administration ofaprotinin in high doses significantly reduces blood loss in connectionwith cardiac surgery, including cardiopulmonary bypass (Bidstrup, et al.(1989) Cardiovasc Surg. 44:640-645)

In developing the constructs, the various fragments comprising theregulatory regions and open reading frame may be subjected to differentprocessing conditions, such as ligation, restriction enzyme digestion,PCR, in vitro mutagenesis, linkers and adapters addition, and the like.Thus, nucleotide transitions, transversions, insertions, deletions, orthe like, may be performed on the DNA which is employed in theregulatory regions or the DNA sequences of interest for expression inthe plastids. Methods for restriction digests, Klenow blunt endtreatments, ligations, and the like are well known to those in the artand are described, for example, by Maniatis et al. (in MolecularCloning: A Laboratory Manual (1989) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.).

During the preparation of the constructs, the various fragments of DNAwill often be cloned in an appropriate cloning vector, which allows foramplification of the DNA, modification of the DNA or manipulation of theDNA by joining or removing sequences, linkers, or the like. Preferably,the vectors will be capable of replication to at least a relatively highcopy number in E. coli. A number of vectors are readily available forcloning, including such vectors as pBR322, vectors of the pUC series,the M13 series vectors, and pBluescript vectors (Stratagene; La Jolla,Calif.).

In order to provide a means of selecting the desired plant cells,vectors for plastid transformation typically contain a construct whichprovides for expression of a selectable marker gene. Marker genes areplant-expressible DNA sequences which express a polypeptide whichresists a natural inhibition by, attenuates, or inactivates a selectivesubstance, including, but not limited to, antibiotic, herbicide etc.

Alternatively, a marker gene may provide some other visibly reactiveresponse, i.e., may cause a distinctive appearance or growth patternrelative to plants or plant cells not expressing the selectable markergene in the presence of some substance, either as applied directly tothe plant or plant cells or as present in the plant or plant cell growthmedia.

In either case, the plants or plant cells containing such selectablemarker genes will have a distinctive phenotype for purposes ofidentification, i.e., they will be distinguishable from non-transformedcells. The characteristic phenotype allows the identification of cells,cell groups, tissues, organs, plant parts or whole plants containing theconstruct.

Detection of the marker phenotype makes possible the selection of cellshaving a second gene to which the marker gene has been linked. Thissecond gene typically comprises a desirable phenotype which is notreadily identifiable in transformed cells, but which is present when theplant cell or derivative thereof is grown to maturity, even underconditions wherein the selectable marker phenotype itself is notapparent.

The use of such a marker for identification of plant cells containing aplastid construct has been described by Svab et al. (1993, supra). Inthe examples provided below, a bacterial aadA gene is expressed as themarker under the regulatory control of chloroplast 5′ promoter and 3′transcription termination regions, specifically the regulatory regionsof the psbA gene (described in Staub et al., EMBO J. (1993)12(2):601-606). Numerous additional promoter regions can also be used todrive expression of the selectable marker gene, including variousplastid promoters and bacterial promoters which have been shown tofunction in plant plastids.

Expression of the aadA gene confers resistance to spectinomycin andstreptomycin, and thus allows for the identification of plant cellsexpressing this marker. The aadA gene product allows for continuedgrowth and greening of cells whose chloroplasts comprise the selectablemarker gene product. Cells which do not contain the selectable markergene product are bleached. Selection for the aadA gene marker is thusbased on identification of plant cells which are not bleached by thepresence of streptomycin, or more preferably spectinomycin, in the plantgrowth medium.

A number of markers have been developed for use with plant cells, suchas resistance to chioramphenicol, the aminoglycoside G418, hygromycin,or the like. Other genes which encode a product involved in chloroplastmetabolism may also be used as selectable markers. For example, geneswhich provide resistance to plant herbicides such as glyphosate,bromoxynil or imidazolinone may find particular use. Such genes havebeen reported (Stalker et al., J. Biol. Chem. (1985) 260:4724-4728(glyphosate resistant EPSP); Stalker et al., J. Biol. Chem. (1985)263:6310-6314 (bromoxynil resistant nitrilase gene); and Sathasivan etal., Nucl. Acids Res. (1990) 18:2188 (AHAS imidazolinone resistancegene)).

Stable transformation of tobacco plastid genomes by particle bombardmentis reported (Svab et. al. (1990), supra) and Svab et al. (1993), supra).The methods described therein may be employed to obtain plantshomoplasmic for plastid expression constructs.

Generally, bombarded tissue is cultured for approximately 2 days on acell division-promoting media, after which the plant tissue istransferred to a selective media containing an inhibitory amount of theparticular selective agent, as well as the particular hormones and othersubstances necessary to obtain regeneration for that particular plantspecies. Shoots are then subcultured on the same selective media toensure production and selection of homoplasmic shoots.

Transplastomic tobacco plants are analyzed for a pure population oftransformed plastid genomes (homoplasmic lines). Homoplasmy is verifiedusing Southern analysis employing nucleic acid probes spanning a regionof the transgene and chloroplast genome (i.e. the insertion region).Transplastomic plants which are heteroplasmic (i.e. contain a mixture ofplastid genomes containing and lacking the transgene) are characterizedby a hybridization pattern of wild type and transgenic bands.Homoplasmic plants show a hybridization pattern lacking the wild typeband.

Alternatively, homoplasmy may be verified using the polymerase chainreaction (PCR). PCR primers are utilized which are targeted to amplifyfrom sequences from the insertion region. For example, a pair of primersmay be utilized in a PCR reaction. One primer amplifies from a region inthe transgene, while the second primer amplifies from a region proximalto the insertion region towards the insertion region. A second PCRreaction is performed using primers designed to amplify the region ofinsertion. Transplastomic lines identified as homoplasmic produce theexpected size fragment in the first reaction, while they do not producethe predicted'size fragment in the second reaction.

Where transformation and regeneration methods have been adapted for agiven plant species, either by Agrobacterium-mediated transformation,bombardment or some other method, the established techniques may bemodified for use in selection and regeneration methods to produceplastid-transformed plants. For example, the methods described hereinfor tobacco are readily adaptable to other solanaceous species, such astomato, petunia and potato.

For transformation of soybean, particle bombardment as well asAgrobacterium-mediated nuclear transformation and regeneration protocolshave been described (Hinchee et al. U.S. Pat. No. 5,416,011, andChristou et al. U.S. Pat. No. 5,015,580). The skilled artisan willrecognize that protocols described for soybean transformation may beused

In Brassica, Agrobacterium-mediated transformation and regenerationprotocols generally involve the use of hypocotyl tissue, a non-greentissue which might contain a low plastid content. Thus, for Brassica,preferred target tissues would include microspore-derived hypocotyl orcotyledonary tissues (which are green and thus contain numerousplastids) or leaf tissue explants. While the regeneration rates fromsuch tissues may be low, positional effects, such as seen withAgrobacterium-mediated transformation, are not expected, thus it wouldnot be necessary to screen numerous successfully transformed plants inorder to obtain a desired phenotype.

For cotton, transformation of Gossypium hirsutum L. cotyledons byco-cultivation with Agrobacterium tumefaciens has been described byFiroozabady et al., Plant Mol. Bio. (1987) 10:105-116 and Umbeck et al.,Bio/Technology (1987) 5:263-266. Again, as for Brassica, this tissue maycontain insufficient plastid content for chloroplast transformation.Thus, as for Brassica, an alternative method for transformation andregeneration of alternative target tissue containing chloroplasts may bedesirable, for instance targeting green embryogenic tissue.

Other plant species may be similarly transformed using relatedtechniques. Alternatively, microprojectile bombardment methods, such asdescribed by Klein et al. (Bio/Technology 10:286-291) may also be usedto obtain nuclear transformed plants comprising the viral single subunitRNA polymerase expression constructs described herein. Cottontransformation by particle bombardment is reported in WO 92/15675,published Sep. 17, 1992. Suitable plants for the practice of the presentinvention include, but are not limited to, soybean, cotton, alfalfa, oilseed rape, flax, tomato, sugar beet, sunflower, potato, tobacco, maize,wheat, rice and lettuce.

The vectors for use in plastid transformation preferably include meansfor providing a stable transfer of the plastid expression construct andselectable marker construct into the plastid genome. This is mostconveniently provided by regions of homology to the target plastidgenome. The regions of homology flank the construct to be transferredand provide for transfer to the plastid genome by homologousrecombination, via a double crossover into the genome. The complete DNAsequence of the plastid genome of tobacco has been reported (Shinozakiet al., EMBO J. (1986) 5:2043-2049). Complete DNA sequences of theplastid genomes from liverwort (Ohyama et al., Nature (1986)322:572-574) and rice (Hiratsuka et al., Mol. Gen. Genet. (1989)217:185-194), have also been reported.

Where the regions of homology are present in the inverted repeat regionsof the plastid genome (known as IRA and IRB), two copies of thetransgene are expected per transformed plastid. Where the regions ofhomology are present outside the inverted repeat regions of the plastidgenome, one copy of the transgene is expected per transformed plastid.The regions of homology within the plastid genome are approximately 1 kbin size. Smaller regions of homology may also be used, and as little as100 bp can provide for homologous recombination into the plastid genome.However, the frequency of recombination and thus the frequency ofobtaining plants having transformed plastids decreases with decreasingsize of the homology regions.

Examples of constructs having regions of homology within the plastidgenome are described in Svab et. al. (1990 supra), Svab et al. (1993supra) and Zoubenko et al. (Nuc Acid Res (1994) 22(19):3819-3824).

As described in more detail in the examples below, constructs aredescribed which provide for enhanced expression of DNA sequences inplant plastids. Various promoter/ribosome binding site sequences areemployed to direct expression in plant plastids.

Promoter sequences of the 16S ribosomal RNA operon (Prrn) are linked toa ribosome binding site (RBS) derived from the T7 bacteriophage gene 10leader sequence (G10L). DNA sequences expressed under the regulatorycontrol of the Prrn/G10L sequence show a significantly higher level ofprotein expression than those levels obtained under the control of otherpromoter/RBS combinations, while expression of mRNA may or may not behigher in these plants.

In the examples below, nucleic acid sequences encoding CP4 EPSP synthase(U.S. Pat. No. 5,633,435) are placed into expression constructs forexpression of EPSP synthase enzyme from the plant plastid. Furthermore,a DNA sequence encoding for hGH (U.S. Pat. No. 5,424,199) is also placedinto expression construct for the expression of human growth hormonefrom the plant plastid. The constructs prepared utilize a ribosomebinding site designed after the T7 bacteriophage gene 10 leader (G10L)to increase the expression of the nucleic acid sequences in the plantplastid.

Plastid expression constructs encoding for the expression of EPSPS andhGH are introduced via a chloroplast transformation vector.

Tobacco lines containing the native encoding sequence to the EPSPSenzyme expressed in plastids under the control of the Prrn/G10Lpromoter/ribosome binding site sequence demonstrate a significantlyhigher level of protein expression than those levels obtained from EPSPSexpressed under the control of the Prrn/rbcL RBS sequence. However,EPSPS mRNA is expressed at a higher level in plants expressing CP4 EPSPSfrom the plastid under the control of the Prrn/rbcL(RBS). These resultsindicate that translation from transcripts containing the T7bacteriophage gene 10 ribosome binding site is more efficient. Inaddition, protein expression levels of EPSPS obtained fromtransplastomic tobacco lines expressing EPSPS under the control of thePrrn/G10L RBS provide for a high level of glyphosate tolerance.

Furthermore, transplastomic tobacco lines transformed to express hGHunder the control of the Prrn/G10L promoter/ribosome binding sitesequence demonstrate a significantly higher level of protein expressionthan those levels obtained from hGH expressed under the control of thePpsbA promoter/RBS sequence.

Increases in protein expression levels of at least approximately 200fold may be obtained from constructs utilizing Prrn/G10L ribosomebinding site for expression of EPSPS and hGH over the expression levelsobtained from other promoter/RBS combinations for plastid expression. Inaddition, protein levels obtained from plastid expression constructsutilizing the Prrn/G10L promoter/RBS sequence may accumulate 50 to 3500fold higher levels than from nuclear expression constructs. Thus,inclusion of the G10L ribosome binding site in plastid expressionconstructs may find use for increasing the levels of protein expressionfrom plant plastids.

Furthermore, the constructs of the present invention may also includesequences to target the expressed protein to a particular suborganellarregion, for example, the thylakoid lumen of the chloroplast. Forexample, as described in the examples below, a nucleotide sequenceencoding a peptide from the plastid genome cytochrome f targets theexpressed aprotinini protein to the thylakoid membrane. Such targetingof expressed proteins may provide for a compartmentalization of theprotein allowing for increased oxidative stability and proper proteinfolding.

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are included forpurposes of illustration only and are not intended to limit the presentinvention.

EXAMPLES Example 1 Expression Constructs

Constructs and methods for use in transforming the plastids of higherplants are described in Zoubenko et al. (Nuc Acid Res (1994)22(19):3819-3824), Svab et al. (Proc. Natl. Acad. Sci. (1990)87:8526-8530 and Proc. Natl. Acad. Sci. (1993) 90:913-917) and Staub etal. (EMBO J. (1993) 12:601-606). Constructs and methods for use intransforming plastids of higher plants to express DNA sequences underthe control of a nuclearly encoded, plastid targeted T7 polymerase aredescribed in U.S. Pat. No. 5,576,198. The complete DNA sequences of theplastid genome of tobacco are reported by Shinozaki et al. (EMBO J.(1986) 5:2043-2049). AU plastid DNA references in the followingdescription are to the nucleotide number from tobacco.

The complete nucleotide sequence encoding the tobacco cytochrome f(petA)is described in Bassham et al, (1991) J Biol Chem 266:23606-23610and Konishi et al. (1993) Plant Cell Physiol 34: 1081-1087.

1A. Promoter/Ribosome Binding Site Sequences

The promoter region of the plastid 16S ribosomal RNA operon (Prrn) islinked to a synthetic ribosome binding site (RBS) patterned on theplastid rbcL gene leader to create the Prrn/rbcLRBS fragment. ThePrrn/rbcLRBS sequence is as described in Svab et al. (1993, supra) forthe Prrn/rbcL(S) fragment.

The promoter region of the plastid psbA promoter (PpsbA) and terminatorsequences (TpsbA) are described in Staub et al. (1993, EMBO J., 12,601-606).

The Prrn/G10L sequence was constructed by annealing two oligonucleotidesequences, T7lead1 and T7lead2 (Table 1), to create the G10L plastidribosome binding site (FIG. 1). The G10L sequence was ligated to the 3′terminus of the Prrn promoter sequence as an EcoRI/NcoI fragment tocreate the Prrn/G10L sequence.

TABLE 1 T7lead1 5′-AAT TGT AGA AAT AAT TTT GTT TAA CTT TAA GAA GGA GATATA CC-3′ T7lead2 5′-CAT GGG TAT ATC TCC TTC TTA AAG TTA AAC AAA ATT ATTTCT AC-3′

Chimeric genes are preferably inserted into the expression vector todirect their transcription from the Prrn promoter. Thus, in the plastidgenome, chimeric genes are transcribed from the Prrn/RBS promoter, orthe Prrn/G10L promoter in the plant plastid.

1B. CP4 EPSPS Plastid Expression Constructs

A plastid expression vector pMON30117 is constructed from a precursorvector pPRV111B (Zoubenko, et al. 1994, supra, GenBank accessionU12813). The vector pMON30117 carries a multiple cloning site forinsertion of a passenger gene in a Prrn/rbcLRBS/Trps16 expressioncassette. The Prrn/rbcLRBS sequence is cloned into pPRV111B vector as anEcoRI/NcoI fragment, and the terminator region from the plastid rps16gene(Trps16) is cloned 3′ of the Prrn promoter as a HindIII/NcoIfragment. The Trps16 fragment comprises the rps116 gene 3′-regulatoryregion from nucleotides 5,087 to 4,939 in the tobacco plasmid DNA.

The pPRV111B backbone of the vector pMON30117 contains a marker gene,aadA, for selection on spectinomycin and streptomycin, and rps 7/12 forthe integration, by homologous recombination, of the passenger DNA intotrnV-rps 7/12 intergenic region.

The plastid expression construct pMON30118 (FIG. 2) was prepared bycloning the native CP4 EPSPS gene fused with the N-terminal five (5)amino acids from the plastid rbcL (described in Svab et al., 1993 supra)gene as an NcoI/SmaI fragment into the multiple cloning site of thevector pMON30117.

The plastid expression construct pMON30123 (FIG. 3) is essentially thesame as pMON30118 with the exception of the deletion of the N-terminalfive (5) amino acids from the plastid rbcL.

The plastid expression construct pMON30130 (FIG. 4) was created byreplacing the native CP4 EPSPS of pMON30123, with a synthetic CP4 gene.This construct also lacks the N-terminal 5 amino acid fusion from theplastid rbcL gene.

The plastid expression construct pMON38773 (FIG. 5) was constructed byreplacing the Prrn/RBS sequence of pMON30123 with the Prrn/G10L promotersequence described above. The EPSPS DNA sequence of pMON38773 also lacksthe N-terminal 5 amino acid fusion from the plastid rbcL gene.

A plastid expression construct, pMON38766 was constructed using thepromoter from T7 phage gene 10 (P-T7), including G10L, CP4 (native) genecoding region, and the terminator sequence from plastid rps16 gene(Trps16).

A plastid expression construct, pMON38797 was constructed using thepromoter from T7 phage gene 10 (P-T7), including G10L, CP4 (synthetic)gene coding region, terminator from plastid rps16 gene (Trps16).

A plastid expression construct, pMON38798 was constructed using thepromoter of the 16SrDNA operon (Prrn), G10L, CP4 (synthetic) gene codingregion, terminator from plastid rps16 gene (Trps16).

A plastid expression construct, pMON38793 was constructed using thepromoter of the 16SrDNA operon (Prrn), a synthetic ribosome binding site(RBS) patterned from the plastid rbcL gene, the glyphosate tolerantPetunia EPSP synthase gene (P-EPSPS, Padgette, et al.(1987) Arch.Biochem. Biophys. 258:564-573) carrying the mutation Glycine to Alanineat amino acid position 101, terminator from plastid rps16 gene (Trps16).

A plastid expression construct, pMON38796 was constructed using thepromoter of the 16SrDNA operon (Prrn), synthetic ribosome binding site(RBS) patterned from the plastid rbcL gene, the glyphosate tolerantAchromobacter (strain LBAA) EPSP synthase gene (U.S. Pat. No. 5,627,061,the entirety of which is incorporated herein by reference) carrying themutation Glycine to Alanine at amino acid position 100, terminator fromplastid rps16 gene (Trps16).

A plastid expression construct, pMON45204, was constructed using thepromoter of the 16SrDNA operon (Prrn) with the G10L, the glyphosatetolerant Pseudomonas (strain LBAA) EPSP synthase gene carrying themutation Glycine to Alanine at amino acid position 100, terminator fromplastid rps16 gene (Trps16).

A plastid expression construct, pMON45201, was constructed using thepromoter of the 16SrDNA operon (Prrn), synthetic ribosome binding site(RBS) patterned from the plastid rbcL gene, wild-type glyphosatetolerant Bacillus subtilis aroE (EPSPS) (U.S. Pat. No. 5,627,061) gene,terminator from plastid rps16 gene (Trps16).

1C. Bucril (bxn) Plastid Expression Constructs

The bxn herbicide resistance gene (U.S. Pat. No. 4,810,648, the entiretyof which is incorporated herein by reference) was removed from theplasmid pBrx47 as an Nco I to Asp718 restriction fragment and clonedinto Nco/Asp7 18 cut pUC120 resulting in plasmid pBrx87. Plasmid pBrx87was then digested with Nco/Xba and cloned into the Nco/Xba sites of theplasmid pLAA21 which contains the Prrn plastid promoter and the rpsL 3′region for plastid expression. The resulting plasmid was designatedpBrx89. Plasmid pBrx89 was digested with Sac I and Hind III and the 1.5kb chimeric bxn gene with plastid expression signals was inserted intothe Sac/Hind III sites of the tobacco plastid homology vector pOVZ44B(Zoubenko et al, Nuc Acids Res 22: 3819-3824 (1994)) to create plasmidpCGN5175.

To construct plasmid pCGN6114, plasmid pBrx90 (a Bluescript plasmidcontaining the bxn gene encoding the bromoxynil specific nitrilase) wasdigested with Nco I/Asc I and the bxn structural gene was substitutedfor the GUS gene in the Nco/Asc digested plasmid pCGN5063 resulting inplasmid pCGN6107. This plasmid contains the bxn gene under the controlof the T7 promoter/gene10 leader at the 5′ end and the psbA/T7 hybridtranscriptional terminator at the 3′ end of the chimeric gene. This T7promoter/bxn chimeric gene was excised from pCGN6107 as a Hind III/Not IDNA segment and moved into the chloromphenical plasmid BCSK+(Stratagene) at the Hind III/Not sites to create plasmid pCGN6109. Thechimeric gene was them moved as a Hind III/Not fragment from pCGN6109into the chloroplast homology vector pOVZ44B described above to createplasmid pCGN6114. Tobacco plants transformed with pCGN6114 require theT7 RNA polymerase be provided in the plant plastid background toactivate transcription of the chimeric bxn gene via the T7 promoter.This system has previously been detailed in McBride et al., PNAS,91:7301-7305 (1994) and McBride et al., U.S. Pat. No. 5,576,198.

1D. BXN/AHAS Plastid Expression Constructs

A plastid expression construct, pCGN5026, is prepared to direct theexpression of BXN and AHAS from the plant plastid. The AHAS nucleotidesequence (described in EP Publication Number 0 525 384 A2, the entiretyof which is incorporated herein by reference) is translationally linkedto the BXN nucleotide sequence (U.S. Pat. No. 4,810,648, the entirety ofwhich is incorporated herein by reference). The AHAS structural geneencoding acetohydroxyacid synthase was cloned from the plasmid pCGN4277as an Nco I to Age DNA fragment into the Nco/Xma sites of plasmid pUC120to create plasmid pCGN5022. This plasmid was then digested with theenzymes BamH I and Pst and a 1.3 kb Bam/Pst DNA segment containing thebxn gene encoding the bromoxynil-specific nitrilase was excised from theplasmid pBrx26 and cloned into the Bam/Pst sites of pCGN5022 to createplasmid pCGN5023. Plasmid pCGN5023 contained a 3.3 kb DNA segmentcontaining the AHAS/bxn operon segment and this fragment. This plasmidwas cut at the unique Pst site and this Pst site was removed andreplaced with a synthetic linker containing a unique Xba I restrictionsite generating plasmid pCGN5024. Plasmid pCGN5024 was digested withNco/Xba and the 3.3 kb Nco/Xba DNA fragment was cloned into the plastidpromoter cassette vector pLAA21(Pst) that had been digested with Nco andXba to remove the GUS gene. The plasmid resulting from this cloning wasdesignated plasmid pCGN5025 and contained the herbicide operon under thecontrol of the plastid promoter Prrn and the rpsL 3′ DNA segment. Theentire chimeric herbicide operon under the control of the plastidexpression elements was excised from pCGN5025 as a Sac I/Pst DNAfragment and cloned into the Sac/Pst sites of the plastid homologycassette vector pOVZ44B (Zoubenko et al, Nuc Acids Res 22:3819-3824(1994)) to facilitate transfer into the tobacco chloroplast genome.

1E. Bt crylAc and bxn Plastid Expression Construct

Plasmid pBrx9 (Stalker and McBride, (1987) J Bacteriol 169:955-960), anoriginal clone from Klebsiella containing a bxn gene DNA segment, wasused as a template to generate an ˜450 bp BamH I/Cla I PCR DNA fragmentthat encompasses the N-terminal end of the bxn gene and includes 44 bpof the 5′ untranslated portion of the native gene. This fragment wasexchanged with the ˜400 bp Bam/Cla fragment in the plasmid pBrx90resulting in plasmid pBrx90.1. This plasmid contains the entire bxn geneand the 44 bp untranslated 5′ DNA segment. The bxn gene was excised fromplasmid pBrx90.1 as a Bam/Asc I DNA segment and inserted into plasmidpCGN5146 at the Bgl II/Asc I sites to generate plasmid pCGN5191. PlasmidpCGN5146 is a pKK233-2 (Pharmacia) derivative containing the full-lengthcryIAc gene encoding the HD-73 Bt protoxin. Plasmid pCGN5191 thencontains the cryIAc and bxn genes in an operon configuration with thebxn gene being the distal gene in the operon. Both genes are under thecontrol of the Ptac promoter for E coli expression in 5191. PlasmidpCGN5191 was digested with Nco/Asc and the Nco/Asc DNA fragmentcontaining the Bt/bxn operon was cloned into the Nco/Asc sites of thechloroplast homology vector pCGN5155, a derivative of pOVZ44B. Theresulting plasmid, pCGN5197 contains the Bt/bxn operon under the controlof the Prrn plastid promoter and rpsL transcription terminator regions.This plasmid facilitated transfer of the Bt/bxn chimeric operon into thetobacco plastid genome.

1F. Phytoene Desaturase Plastid Expression Constructs

The crtI gene was obtained as a Hind III/Sal I PCR fragment from theoriginal plasmid containing the Erwinia carotova crt operon (Misawa etal, (1994) Plant Jour 6:481-489)) and cloned as a Hind III/Sal DNAsegment into BCSK+ (Stratagene) at the Hind III/Sal sites to generateplasmid pCGN5172. The crtI fragment was cloned from pCGN5172 as an NcoI/Sal I fragment into pCGN5038 (a derivative of pOVZ44B) to create theplastid expression construct pCGN5177. This construct directs theexpression of the crtI sequence from the Prrn promoter and the rps16terminator sequence. This plasmid facilitated the transfer of thechimeric crtI gene into the tobacco plastid genome.

1G. hGH Expression Constructs for Plant Transformation

Nuclear Expression Constructs

The construct pWRG4747 (FIG. 6) was constructed to direct the expressionof hGH in the plant nuclear genome. This vector contains the hGHoperably linked to the Figwort Mosaic Virus promoter (U.S. Pat. No.5,378,619, the entirety is incorporated herein by reference) and theCTP2 leader for directing the hGH protein into the plastid. TheFMV/CTP2L::hGH::NpA fragment is cloned along with the DNA sequenceconferring resistance to Kanamycin between the right and left borders(RB and LB) of the transfer DNA (TDNA) of Agrobacterium tumefaciens todirect the integration into the nuclear genome.

The nuclear transformation vector pWRG4744 contains essentially the sameelements as pWRG4747 except the construct lacks the CTP2 leader and thehGH protein is directed to the plant cell cytoplasm.

Plastid Expression Constructs

The plastid expression vector pWRG4838 (FIG. 7) was constructed usingthe full length hGH gene expressed from the promoter and terminatorregion from the psbA gene, PpsbA and TpsbA, respectively (described inStaub et al. (1993), supra). This chimeric promoter-gene-terminatorfusion (PpsbA::hGH::TpsbA) is cloned adjacent to the selectable markergene aadA also driven by the plastid expression elements of the psbAgene. The two chimeric gene sequences are cloned into a vector betweentwo sequences which direct the integration of the chimeric genesequences into the tobacco plastid genome upstream of the plastid16SrDNA. This is joined to a 1 kb Ampicillin resistance gene whichprovides for selection of E. coli containing the construct and the pUCorigin of replication for plasmid maintenance in E. coli.

The plastid expression construct pMON38755 (FIG. 8) was prepared usingthe hGH DNA sequence translationally fused at the N-terminus with theyeast ubiquitin gene (Ozkaynak, et al. (1984) Nature 312:663-666),creating the Ubi-hGH fusion gene. The Ubi-hGH fusion gene is cloned nextto the aadA gene for selection of transplastomic tobacco on mediacontaining spectinomycin or streptomycin (from pPRV112B described inZoubenkoet al. (1994) supra). Sequences are included for the homologousrecombination of sequences encoding for hGH and aadA expression. Thesesequences are obtained from the vector pPRV112B described in Zoubenko etal. (1994, supra). These sequences are joined to a 1 kb ampicillinresistance gene which provides for selection of E. coli containing theconstruct and the pUC origin of replication for plasmid maintenance inE. coli.

The plastid expression construct pMON38794 (FIG. 9) contains essentiallythe same elements as pMON38755, with the exception that the 0.15 kb psbApromoter sequence is replaced with the Prrn/G10L promoter sequencedescribed above.

1H. Constructs for the Expression of Aprotinin in Plastids

A series of constructs were prepared to direct the expression of thepharmaceutical protein aprotinin from the plastid. The nucleic acidsequence encoding for aprotinin (FIG. 10) was cloned into a plastidexpression construct to control the expression of aprotinin from the T7gene 10 leader promoter which is induced from a nuclearly expressed,plastid targeted T7 Polymerase. The constructs used in which theaprotinin sequence was cloned are as described in U.S. Pat. No.5,576,198, the entirety of which is incorporated herein by reference.The plastid transformation vector pCGN6146 is designed by replacing theDNA sequence encoding for GUS from pCGN4276 (described in U.S. Pat. No.5,576,198) with the coding sequence of aprotinin. The tobacco plastidtransformation construct pCGN6147 contains the same elements as pCGN6146except pCGN6147 contains the six 5′ amino acids of the GUS encodingsequence ligated to the 5′ terminus of the aprotinin encoding sequence.The six amino acids of the 5′ terminus of the GUS nucleotide sequenceare included to aid in the translation of the aprotinin protein. Thetobacco plastid transformation vector pCGN6156 is essentially the sameas pCGN4276 except the coding region of aprotinin is cloned to the 3′end of the GUS coding sequence. Thus, pCGN6156 contains as operablylinked the T7 promoter, a DNA sequence encoding for GUS fused with theDNA sequence encoding for aprotinin and the psbA 3′ transcriptiontermination sequence.

A plastid expression construct, pCGN6154, was constructed from pCGN4276by replacing the GUS coding sequence with the aprotinin protein operablylinked to the 3′ terminus of the coding sequence of cytochrome f (peta)of the tobacco chloroplast. Thus, pCGN6154 contains the T7 promotersequence operably linked to the nucleotide sequence of peta andaprotinin. The peta sequence is included to direct the expressedaprotinin protein to the thylakoid.

Example 2 Plant Transformation

2A. Nuclear Transformation

Tobacco plants transformed to express the constructs pWRG4744 andpWRG4747 in the nucleus of a plant cell may be obtained as described byHorsch et al. (Science (1985) 227:1229-1232).

2B. Plastid Transformation

Tobacco plastids are transformed by particle gun delivery ofmicroprojectiles as described by Svab and Maliga (Proc. Natl. Acad. Sci.(1993) 90:913-917), and described herein.

Dark green, round leaves are cut, preferably from the middle of theshoots, from 3-6 week old Nicotiaria tabacum cv. Havana which have beenmaintained in vitro on hormone free MS medium (Murashige and Skoog,(1962) Physiol Plant. 15,473-497) supplemented with B5 vitamins inPhytatrays or sundae cups with a 16 hour photoperiod at 24° C. Each cutleaf is then placed adaxial side up on sterile filter paper over tobaccoshoot regeneration medium (TSO medium: MS salts, 1 mg/1N⁶-benzyladenine, 0.1 mg/l 1-naphthaleneacetic acid, 1 mg/l thiamine,100 mg/l inositol, 7 g/l agar pH 5.8 and 30 g/l sucrose). Leaves arepreferably placed in the center of the plate with as much contact withthe medium as possible. The plates are preferably prepared immediatelyprior to use, but may be prepared up to a day before transformation byparticle bombardment by wrapping in plastic bags and storing at 24° C.overnight.

Tungsten or gold particles are sterilized for use as microcarriers inbombardment experiments. Particles (50 mg) are sterilized with 1 ml of100% ethanol, and stored at −20° C. or −80° C. Immediately prior to use,particles are sedimented by centrifugation, washed with 2 to 3 washes of1 ml sterile deionised distilled water, vortexed and centrifuged betweeneach wash. Washed particles are resuspended in 500 μl 50% glycerol.

Sterilized particles are coated with DNA for transformation. Twenty-fivemicroliter aliquots of sterilized particles are added to a 1.5 mlmicrofuge tube, and 5 μg of DNA of interest is added and mixed bytapping. Thirty-five microliters of a freshly prepared solution of 1.8MCaCl₂ and 30 mM spermidine is added to the particle/DNA mixture, mixedgently, and incubated at room temperature for 20 minutes. The coatedparticles are sedimented by centrifuging briefly. The particles arewashed twice by adding 200 μl 70% ethanol, mixing gently, andcentrifuging briefly. The coated particles are resuspended in 50 μl of100% ethanol and mixed gently. Five to ten microliters of coatedparticles are used for each bombardment.

Transformation by particle bombardment is carried out using the PDS 1000Helium gun (Bio Rad, Richmond, Calif.) using a modified protocoldescribed by the manufacturer. Plates containing the leaf samples areplaced on the second shelf from the bottom of the vacuum chamber andbombarded using the 1100 p.s.i. rupture disk. After bombardment,petriplates containing the leaf samples are wrapped in plastic bags andincubated at 24° C. for 48 hours.

After incubation, bombarded leaves are cut into approximately 0.5 cm²pieces and placed abaxial side up on TSO medium supplemented with 500μg/ml spectinomycin. After 3 to 4 weeks on the selection medium, small,green spectinomycin resistant shoots will appear on the leaf tissue.These shoots will continue to grow on spectinomycin containing mediumand are referred to as primary putative transformants.

When the primary putative transformants have developed 2 to 3 leaves, 2small pieces (approximately 0.5 cm²) are cut from each leaf and used foreither selection or for a second round of shoot regeneration. One pieceis placed abaxial side up on plates containing TSO medium supplementedwith 500 μg/ml spectinomycin, and the other piece is placed abaxial sideup on TSO medium supplemented with 500 μg/ml each of spectinomycin andstreptomycin. Positive transformants are identified as the shoots whichform green callus on the TSO medium containing spectinomycin andstreptomycin.

After 3 to 4 weeks, the tissue placed on TSO medium containing onlyspectinomycin, which has been identified as positive on the TSO mediumwith spectinomycin and streptomycin, will develop green shoots. Two tofour shoots of each positive transformant are selected and transferredto TSO medium supplemented with 500 μg/ml spectinomycin for generationof roots. Southern analysis is performed on 2 shoots to confirmhomoplasmy as described below. Shoots from homoplasmic events aretransferred to the greenhouse for seed production, while transformantswhich are not homoplasmic are sent through a second round orregeneration on TSO medium with 500 μg/ml spectinomycin to attainhomoplasmy.

Example 3 Analysis of Transplastomic Tobacco Plants Transformed withHerbicide Tolerance Constructs

3A. Southern Analysis

Transformed plants selected for marker aadA marker gene expression areanalyzed to determine whether the entire plastid content of the planthas been transformed (homoplasmic transformants). Typically, followingtwo rounds of shoot formation and spectinomycin selection, approximately50% of the transgenic plantlets which are analyzed are homoplasmic, asdetermined by Southern blot analysis of plastid DNA. Homoplasmicplantlets are selected for further cultivation.

Genomic DNA is isolated from transformed tobacco plants,electrophoresed, and transferred to filters as described in Svab et al.((1993), Proc Natl Acad Sci, 90:913-917).

Homoplasmic tobacco plants transformed to express CP4 EPSPS in plastidswere identified using a probe prepared from a 2.4 kb EcoRI/EcoRVfragment from the vector pOVZ2 (similar to pOVZ15 described in Zoubenko,et al. 1994, supra). The 2.4 kb probe fragment encompasses part of thetargeting sequence.

Results of the Southern hybridizations identified 3 homoplasmic linesfrom tobacco transformed with the constructs pMON30123 and pMON30130 and1 line from tobacco transformed with pMON38773 for further analysis.

The complete disappearance of the 3.27 Kb native tobacco BamHI fragmentin the lines 30123-19-1A, 30123-23-2A, 30123-18-1B, 30130-51-2A,30130-57-1P, and 38773-6 with a probe covering the region ofintegration, and the appearance of expected sized bands for the insertedDNA fragments in those transformants, 5.14 kb and 0.9 kb, establishesthat the transformed plants are homoplasmic for the intended constructs.

Results of the Southern hybridizations identified 3 homoplasmic linesfrom tobacco transformed with pCGN5177, lines 74-1B-P, 74-2 and 74-7.

Transplastomic 5175 and 6114 tobacco lines were analyzed by Southernhybridization for homoplasmy as described above. Results of the Southernhybridizations identified 4 homoplasmic lines from tobacco transformedwith pCGN6114.

Results from hybridizations of 5175 transplastomic tobacco linesidentified one line, 76-4A-F, as homoplasmic, and a second line as 95%homoplasmic.

Homoplasmic tobacco plants transformed to express BXN/AHAS in plastidswere identified using Southern hybridizations as described above.

Results of the Southern hybridizations identified 14 homoplasmic linesfrom tobacco transformed with pCGN5026. The filters were reprobed with aBXN gene fragment, and 21 lines were found to contain BXN, 14 lines ofwhich were homoplasmic.

3B. Northern Analysis

In order to determine the level of transcription of the EPSPS, BXN orAHAS mRNA expressed in the transplastomic tobacco plants, Northern blothybridizations were performed with total RNA isolated from each of thelines identified. Total RNA was isolated using TRIzol reagent (Gibco-BRLLife Technologies, Gaithersburg, Md.) according to the manufacturersprotocol. Total RNA, 2 μg, was separated on a denaturing agarose gel andtransferred to nylon membrane (Maniatis et al., 1989, supra).Radioactive probes for hybridizations were prepared using random primerlabeled (using Random Primer labeling kit from Boehringer Mannheim) CP4EPSPS, phytoene desaturase, BXN, or AHAS fragments and hybridizationswere carried out in 2×SSPE (Maniatis, et al., 1989,supra), at 60° C.Filters were stripped and reprobed with a plastid 16S ribosomal RNA geneprobe (from pPRV112A, Zoubenko, et al., 1994, supra) to confirmhomogenous loading of RNA on the filter.

Results of the Northern hybridizations performed with EPSPS probesdemonstrate that all seven (7) lines examined express CP4 EPSPS mRNA.Hybridizations performed with the 16S ribosome probe confirm thatdenaturing gels were loaded with similar amounts of total RNA for eachsample. Furthermore, transplastomic tobacco lines expressing EPSPS fromthe Prrn/rbcL(RBS) (pMON30123) regulatory elements express EPSPS mRNA tohigher levels than tobacco plants homoplasmic for EPSPS controlled bythe Prrn/G10L (pMON38773) promoter/RBS sequences.

Results of Northern hybridizations performed with BXN, AHAS and crtIprobes demonstrates that all homoplasmic 5026, 5175, and 5177 tobaccolines expressed crtI, BXN and/or AHAS mRNA.

3C. Western Blot Analysis of Tobacco CP4 EPSPS

To determine the expression of the EPSPS, Western blot analysis wasperformed on a single line from each construct, pMON30123, pMON30130,and pMON38773.

Total soluble protein was extracted from frozen leaf tissue by grinding250 mg tissue in 250 μl of PBS buffer (1 mM KH₂PO₄, Na₂HPO₄, 0.137MNaCl, 2.7 mM KCl pH 7.0) containing protease inhibitors. The homogenateis centrifuged for 5 minutes, and the supernatant is transferred to afresh tube. The concentration of the protein in the supernatant isdetermined using a protein concentration assay (BioRad, Richmond,Calif.).

Extracted total protein is electrophoresed on a 4-20% SDS-PAGE gel(Sigma, St Louis, Mo.), and transferred to PVDF membrane in 1× SDS-PAGEbuffer (Maniatis et al 1989, Cold Spring Harbor Press). Standards ofquantitated purified CP4 EPSPS protein were used to quantify theexpression of the CP4 EPSPS as expressed in the plant plastid.

Western hybridizations are performed as described in Staub and Maliga(1993) EMBO Journal, 12(2) 601-606, except using antibodies raised toEPSPS. PVDF membranes containing the transferred electrophoresed proteinwere incubated in a blocking solution of PBS buffer containing 0.05%Tween-20 (PBS-T) and 5% milk overnight at 4° C. The membranes are thenincubated in a solution of PBS-T containing 1% milk and a primaryantibody raised in goats to the CP4 EPSPS for 2 hours at roomtemperature. The membranes are washed three times in a solution of PBS-Tcontaining 0.1% milk, each wash for 5 minutes at room temperature. Themembranes are then incubated in a solution of PBS-T containing 1% milkand sheep anti-goat antibody for 1 hour at room temperature, and washedagain in PBS-T containing 0.1% milk, three times for 10 minutes at roomtemperature. A final wash using only PBS-T is performed beforedeveloping the membranes using a nonradioactive detection kit (ECL,Amersham).

TABLE 2 Construct Number Event Number % Total Soluble Protein pMON30123T18-23-2A 0.001 pMON30130 T18-51-2P 0.002 pMON38773 9706-6-1 0.2

The results listed in Table 2 demonstrate that significant increases inthe level of EPSPS protein may be obtained from plants transformed toexpress EPSPS from the Prrn/G10L promoter. These results demonstratethat EPSPS expression driven by the Prrn/rbcLRBS regulatory sequencesmay produce approximately 0.001% of the total soluble protein as EPSPS,while in plants expressing EPSPS from the Prrn/G10L regulatory sequencesexpress 0.2% of the total soluble protein as EPSPS. Subsequent lineshave demonstrated total soluble protein of about 1% EPSPS when expressedfrom the Prm/G10L regulatory sequences. These results, taken togetherwith the results of the Northern hybridizations above, indicate thatmore efficient translation may be obtained from the G10L ribosomebinding site.

Western immunoblot hybridization were also performed on 2 homoplasmic5026 tobacco lines as described above, using antibodies raised againstbromoxynil. The results of Western immunoblot analysis of total solubleprotein extracted from tobacco lines transformed with pCGN5026demonstrated that both homoplasmic lines produced nitrilase protein.

Western immunoblot analysis was performed as described above from totalprotein extracted from tobacco lines transformed with pCGN6114 andpCGN5197.

The results of the analysis demonstrated that bromoxynil was produced in6114 tobacco lines ranging from 1% to 2% of the total soluble leafprotein.

The results of the Western analysis of the 20 5197 tobacco linesdemonstrated that bromoxynil and Bt were both produced as 1% of thetotal soluble leaf protein.

3D. Analysis of EPSPS Enzyme Activity

The EPSPS enzyme activity in transplastomic tobacco plants containingthe plastid expression vector pMON38773 was determined using a highpressure liquid chromatography (HPLC) assay.

Methods for the analysis of EPSPS enzyme activity are described inPadgette et al. (J. Biol. Chem. (1988)263:1798-1802 and Arch. Biochem.Biophys. (1987)258:564-573) and Wibbenmeyer et al. (Biochem. Biophys.Res. Commun. (1988)153:760-766). The results are summarized in Table 3below.

TABLE 3 Nuclear Nuclear Enzymatic Activity % Total Plants ChloroplastRange In Range 38773-6 1–3.7 μmol/mg 1% 16.39 nmol/mg >0.1 μmol/mg16% >10 nmol/mg 55% >1 nmol/mg 32% 0 nmol/mg 3% These resultsdemonstrate that EPSPS expression in plastids produces active EPSPSenzyme.3E. Analysis for Glyphosate Tolerance

A transplastomic tobacco line homoplasmic for the construct pMON38773was tested in vitro to determine the highest level of glyphosatetolerance. Explant tissue was prepared from leaf pieces of nontransgenicwild type tobacco control, Havanna, plants and the homoplasmic tobaccoline 38773-6 and cultured for regeneration of shoots on TSO medium(described above) supplemented with glyphosate levels of 50 μM, 75 μM,100 μM, 150 μM and 200 μM. The results are summarized in Table 4 below.The number of explants producing shoots was determined at 3 weeks and 6weeks after explant preparation and culturing on glyphosate containingmedium.

TABLE 4 Total Number Number Glyphosate Number Regenerating Regenerating% Explant Level (μM) Explants 3 Weeks 6 Weeks Regeneration Wild Type  5010 0 0 0  75 10 0 0 0 100 10 0 0 0 150 10 0 0 0 200 10 0 0 0 38773-6  508 5 8 100  75 18 14 18 100 100 17 12 15 88 150 18 10 16 89 200 16 8 1586

The above results demonstrate that at all levels of glyphosate examined,shoots regenerated from explants prepared from a tobacco linehomoplasmic for pMON38773, while no shoots regenerated from explantsprepared from nontransformed control plants. These results suggest thattobacco plants expressing EPSPS in plastids demonstrate tolerance toglyphosate levels of at least 200 μM.

Additional transplastomic lines were tested in vitro for glyphosatetolerance as bed above. The results are shown in Table 5.

TABLE 5 Summary of tobacco plastid transformation experiments withvarious constructs containing EPSPS genes. No. of shoots ConstructSpec/strep (+) Gly 50 uM(+) pMON38766 (Wild) 1 0 pMON38766 (T7) 6 0pMON38773 (Wild) 9 5(1) pMON38797 (Wild) 2 0 pMON38798 6 6 pMON38793 8 0pMON38796 4 0 pMON45201 9 3 pMON45204 12 * (No. of shoots positive at 1mM glyphosate)

These results demonstrate that these transplastomic lines show toleranceto glyphosate.

The numbers in parentheses are the number of shoots resistant toselection at 1 mM glyphosate. Thus, as can be seen in table 5, tobaccolines are generated that are tolerant of selection at 1 mM glyphosate.

Homoplasmic tobacco plants of the line 38773-6 are sprayed withglyphosate using a track sprayer at concentrations corresponding to 0oz/acre, 16 oz/acre, 32 oz/acre and 64 oz/acre to test for whole planttolerance. Plant height was measured before and after spraying withglyphosate. The vegetative injury data was collected two weeks afterspraying, while the reproductive injury data was collected at plantmaturity.

Initial results indicate that homoplasmic tobacco lines sprayed aretolerant of glyphosate at the concentration of 16 oz/acre asdemonstrated in the vegetative tissue injury (Table 6). As can be seenin Table 5 transplastomic lines were generated which demonstrated a goodlevel of glyphosate tolerance at 32 oz/Acre. In subsequent experimentswith additional transformed lines, transplastomic lines have showntolerance to glyphosate at a level of 64 oz/Acre.

Tolerance is characterized by the continued growth and greening oftissues sprayed with glyphosate. However, as the concentration ofglyphosate applied increased, there was a corresponding increase in thelevel of vegetative injury. In contrast, nontransformed control plantswhich were highly susceptible to glyphosate concentrations as low as 16oz/Acre.

TABLE 6 Roundup Plant height Plant height (cm) Vegetative Fertility rate(cm) before after spray injury rating Plant No. Construct (oz/A) spray(Mar. 19, 1998) (Apr. 3, 1998) (Apr. 6, 1998) (Jun. 12, 1998) 1 38773 012.2 30.5 0 0 2 38773 0 13.6 34.0 0 0 3 38773 0 8.6 23.8 0 0 4 38773 08.6 26.2 0 0 5 38773 0 7.8 28.8 0 0 6 38773 0 12.8 31.5 0 0 7 38773 012.2 31.6 0 0 8 38773 0 11.6 35.5 0 0 9 38773 16 9.0 29.0 1 0 10 3877316 14.4 31.0 0 0 11 38773 16 13.4 32.0 0 0 12 38773 16 13.2 30.0 0 0 1338773 16 14.2 30.5 0 1 14 38773 16 14.0 33.0 0 0 15 38773 16 13.2 30.2 00 16 38773 16 14.9 30.4 0 0 17 38773 32 12.0 26.5 2 4 18 38773 32 11.625.4 1 1 19 38773 32 9.4 22.0 1 3 20 38773 32 11.2 23.0 2 4 21 38773 3213.8 25.8 1 2 22 38773 32 12.4 23.0 1 4 23 38773 32 10.2 19.0 2 4 2438773 32 13.8 23.2 2 3 26 38773 64 11.8 20.0 2 5 27 38773 64 13.0 22.0 25 28 38773 64 12.2 18.0 3 5 29 38773 64 15.8 23.0 2 5 30 38773 64 10.417.5 2 5 32 38773 64 15.0 18.5 2 5 33 38773 64 13.8 21.8 2 5 34 38773 6413.6 19.0 3 5 35 38773 64 10.8 16.0 3 5 36 Wild type 0 21.0 40.6 0 0 37Wild type 0 16.0 38.0 0 0 38 Wild type 0 15.0 34.6 0 0 39 Wild type 017.6 32.2 0 0 40 Wild type 0 15.0 31.6 0 0 41 Wild type 0 14.0 32.0 0 042 Wild type 16 10.0 11.8 3 5 43 Wild type 16 8.0 10.0 3 5 44 Wild type16 8.6 11.0 3 5 45 Wild type 16 8.0 14.0 3 5 46 Wild type 16 9.8 11.0 35 47 Wild type 16 10.4 14.0 3 5 48 Wild type 32 10.8 13.2 3 5 49 Wildtype 32 9.0 13.0 3 5 50 Wild type 32 8.0 10.2 3 5 51 Wild type 32 11.014.0 4 5 52 Wild type 32 9.8 13.0 3 5 53 Wild type 32 8.0 10.8 4 5 54Wild type 64 7.5 8.6 4 5 55 Wild type 64 11.2 12.5 4 5 56 Wild type 6410.2 12.8 4 5 57 Wild type 64 11.5 13.0 4 5 58 Wild type 64 13.0 15.0 45 59 Wild type 64 9.8 11.2 4 5 Vegetative injuries: 0 = normal plant 1 =slight chlorosis of new leaves and stunting 2 = severe chlorosis of newleaves, malformation of new leaves, and severe stunting 3 = dying plant4 = dead plant Fertility ratings: 0 = Fertile, no delay in maturity,lots of seed 1 = Some abortion, slight delay in seed set, seed 2 =Significant abortion, significant delay in seed set, some seed 3 = Verysevere abortion, immature seed pots, a few seed 4 = malformed flowers;if flowered, extreme delay in flowering and no seed produced 5 = deadplant3F. BT/BXN Analysis

Homoplasmic tobacco plants of the lines 5175 and 5197 are sprayed withBuctril herbicide at a concentration of 4% to test for whole planttolerance.

Results of the spray test with Buctril demonstrated that all 5197 linesexpressing bxn were completely resistant when sprayed with a solutioncontaining 4% Buctril herbicide.

Two lines out of six 5175 lines tested were completely resistant to theherbicide when sprayed with a 4% solution containing Buctril.

3G. Norflurazon Resistance Analysis

An experiment was set up to determine the efficacy of the Crt I traitwith respect to resistance to the herbicide Norflurazon. Three 5177transformed lines, 74-1B-P, 74-2-A, and 74-7-C and three control lineswere planted. Plants were grown for seven weeks and then watered with a3 μM Norflurazon solution. Plants negative for the presence of the crtIplastid-borne gene were bleached by Norflurazon treatment, positiveplants stayed green and continued to grow.

The results show that the three homoplasmic 5177 tobacco lines wereresistant to the 3 μM Norflurazon solution, while the control plantswere all susceptible to the solution (Table 7).

TABLE 7 Line Control/Transgenic Result Xanthi Control Susceptible 2560AXanthi Control Susceptible 75-5D-A Control Susceptible 74-1B-Phomoplasmic Resistant 74-2-A homoplasmic Resistant 74-7-C homoplasmicResistant

Example 4 Analysis of hGH Transgenic Tobacco Plants

4A. Southern Analysis

Transformed plants selected for aadA marker gene expression are analyzedto determine whether the entire plastid content of the plant has beentransformed (homoplasmic transformants). Homoplasmic plants are selectedusing Southern hybridization for further cultivation.

Genomic DNA is isolated from transformed tobacco plants,electrophoresed, and transferred to filters as described in Svab et al.((1993), Proc Natl Acad Sci, 90:913-917).

Homoplasmic tobacco plants transformed to express hGH were identifiedusing a probe prepared from a 2.4 kb EcoRI/EcoRV fragment from thevector pOVZ2 (similar to pOVZ15 described in Zoubenko, et al. 1994,supra). The 2.4 kb probe fragment encompasses part of the targetingsequence.

The complete disappearance of the 3.27 Kb native tobacco BamHI fragmentin the lines with a probe covering the region of integration, and theappearance of the expected size band for the inserted DNA fragments inthose transformants, 5.6 kb, establishes that the transformed plants arehomoplasmic for the intended constructs.

4B. Protein Expression Analysis

Homoplasmic tobacco lines expressing hGH and nuclear tobaccotransformants are used to determine the expression of the hGH protein.Western blot analysis was performed on tobacco lines containingconstructs pWRG4838, pMON38755 and pMON38794 for plastid expression andan ELISA assay was used for transgenic tobacco lines containing pWRG4744and pWRG4747 for nuclear expression of hGH.

Total protein extractions and western blot procedures were performed asdescribed above, with the exception of the primary antibody was raisedagainst hGH.

TABLE 8 Expression Levels of hGH in Tobacco Nuclear Genome and Plastidgenome Expression Level Construct Expression % Total Soluble ProteinpWRG4744 nuclear 0.002–0.125% pWRG4747 nuclear 0.002–0.025% pWRG4838plastid 0.2% pMON38755 plastid 1.0% pMON38794 plastid 7.0%

Results of the Western analysis (Table 8) demonstrates that hGHexpressed in plastids of plant cells accumulates to significantly higherlevels than hGH expressed in the nucleus and targeted to either thecytoplasm or plastid of plant cells. Tobacco plants transformed toexpress hGH in the nucleus accumulated hGH levels of 0.002% (cytoplasmictargeted) to 0.025% (plastid targeted) of total soluble leaf protein,while tobacco plants expressing hGH in the plastid accumulated hGHlevels of 0.2% to 7.0% of the total soluble leaf protein as hGH.Furthermore, homoplasmic tobacco plants expressing hGH directed from thePrrn/G10L regulatory sequences accumulate 35 fold higher levels of hGHthan homoplasmic tobacco plants expressing hGH directed from the PpsbApromoter sequence. The higher level of expression may be due to thestrong Prrn promoter and/or to enhanced translation of the fusion genemediated by the gene 10 leader rbs region. Leaves of different ages hadvaried hGH accumulation patterns, with mature and old leaves havingsimilar levels and younger leaves much less hGH. This is consistent withthe lower chloroplast number in young leaves.

Interestingly, both ubiquitin-hGH and processed hGH accumulated in thepost-harvest extracts of the Nt-38755 and Nt-38794 lines. Ubiquitinprocessing was often observed at >50% of total hST protein species,depending on extraction conditions. This result confirms the utility ofthe fusion protein approach in chloroplast-expressed proteins. Theappearance of an extra band observed in the Nt4838 sample is consistentwith an hGH dimer.

For comparison of expression systems in plants, nuclear transgenicplants were generated that express hGH from two different sets ofexpression signals. The wrg4747 and wrg4776 constructs expresses hGHusing the strong Figwort Mosaic Virus promoter or the Cauliflower MosaicVirus 35S promoter, respectively. The wrg4747 construct employs achloroplast transit peptide to post-translationally target hGH tochloroplasts (FMV::CTP-hGH), whereas the wrg4776 construct targets thehGH through the endoplasmic reticulum (ER) to the secretory pathway(35S::ER-hST). Transgenic lines for both constructs were obtainedthrough particle bombardment. Expression of hST was quantitated by ELISAassay and shown to be less than 0.025% tsp. This level of expression isat least 300-fold lower than the pMON38794 lines, proving thefeasibility of the chioroplast expression system for the potentialproduction of hST.

4C. Characterization of hGH Protein Expressed in the Plastid

In order to determine whether the hGH expressed from plastids wasproperly processed, experiments were performed to determine correctfolding and bioactivity.

Two bottom leaves of transplastomic tobacco lines containing pMON38794were used to extract and purify hGH. Large veins were removed from theexcised leaves, and the leaf tissue was cut into small sections(approximately 0.5 cm²). The leaf pieces were flash frozen in liquidnitrogen and ground to a fine powder in a chilled mortar and pestle. Tengrams of frozen, ground leaf tissue was added to ice cold 100 mM Trisbase solution (30 ml) and mixed vigorously by vortexing for 5 minutes.The solution was filtered through a single layer of cheese cloth.

From the filtered solution, three separate samples were prepared. Thefirst sample was prepared by centrifuging 4 ml of the filtrate for 1minute at 16,000 rpm. The centrifugate was aliquoted into 1 ml vials andfrozen in dry ice. The remaining filtrate was centrifuged for 10 minutesat 4800 rpm, and several 0.5 ml aliquots were frozen as above for thesecond sample.

To the remaining centrifuged filtrate (approximately 25 ml), 200 μl ofglacial acetic acid was added to lower the pH from 8.2 to 4.56. Thesolution was centrifuged at 4800 rpm for 30 minutes, and the supernatantwas frozen over dry ice for the third sample.

Total soluble protein (TSP, Table 9) was calculated in these samples bystandard protein assay procedures (Maniatis,), and the percent purity ofhGH was calculated based on results from Western blot analysis usingknown concentrations of starting material.

TABLE 9 TSP GP2000 Sample ID mg/mL mg/L % Purity Filtered Extractimmediately centrifuged 6.3 28 0.45% and frozen Filtered extractcentrifuged at 4800 rpm 6.4 28 0.45 for 10 min and frozen pH adjustedand centrifuged extract 0.75 21  2.8%

The pH adjusted and centrifuged extract was purified by ReversePhase-HPLC (RP-HPLC) for electrospray mass spectrometry andamino-terminal amino acid sequencing. RP-HPLC was performed using aPerkin-Elmer series 200 pump and autosampler and a Vydac C8 (250 by 4.6mm) RP-HPLC column. 750 microliters of sample was loaded onto the columnequilibrated with 20 mM trifluoroacetic acid (TFA) and 50% acetonitrile.After loading, the column was washed for 2 minutes with 50%acetonitrile, 20 mM TFA followed by a 2% linear acetonitrile gradientover 10 minutes followed by a 10% acetonitrile gradient over 1 minute.The flow rate was a constant 1.5 ml/minute with the column eluatemonitored at 278 nm with a Perkin-Elmer 785 detector. Data was collectedand analyzed with a PE-Nelson Turbochrom data system.

The results of the RP-HPLC analysis are shown in FIG. 11. Peak I(tallest peak) has the retention time expected for properly folded,native 22 kDa GP2000. This peak was collected and dried down in a SavantSpeed-Vac for amino terminal sequencing and electrospray massspectrometry.

Electrospray ionization mass spectrometry (MS) analysis used a MicromassQ-Tof electrospray time-of-flight mass spectrometer. The samples wereprepared by resuspending in 50% methanol +2% acetic acid, and infusedinto the source of the mass spectrometer at a rate of 4 mL/min. The rawdata shown in FIG. 12 shows a series of ions corresponding to thespecie(s) present in the sample with varying numbers of protonsattached. The axes of this spectrum are intensity versus mass-to-chargeratio of the specie(s) present. A deconvolution algorithm is used toconvert this series of multiply charged ions into a molecular weightspectrum.

The results of the mass spectrometry of the RP-HPLC peak I shows 4 majorprotein species of different molecular mass. The 21,997 kDa speciesrepresents the predicted mass of hGH with the predicted N-terminal Pheremoved by over-cleavage of the Ubiquitin protease with an N-terminalproline residue (P-hGH). The 22,124 kDa species represents the predictedmass of properly processed, correct amino acid sequence of hGH havingthe N-terminal phenylalanine (F-hGH). The 22,507 kDa and 22,664 kDaspecies are thought to represent an hGH with the N-terminal Phe and hGHwhich has been modified during plant extraction procedures,respectively. The calculated molecular mass of the proteins suggeststhat the hGH expressed from the plastid is properly folded (i.e. thecorrect disulfide bonds are created).

Equivalent mobility to refolded E. coli produced protein indicatesformation of the two disulfide bonds and proper folding of thechloroplast derived hGH. This result was surprising because of theprokaryotic nature of chloroplasts. There are no known,plastid-expressed proteins that have disulfide bonds. However,nuclear-encoded, imported enzymes can be activated by disulfide bondoxidation/reduction cycles, presumably using the chloroplast thioredoxinsystem (Jacquot, et al. (1997) New Phytol. 136:543-570) or a recentlydiscovered chloroplast protein disulfide isomerase (Kim and Mayfield(1997) Science 278: 1954-1957). This result suggests that theprokaryotic organelle has the machinery needed to fold complexeukaryotic proteins in the soluble chloroplast stroma compartment. Thisis distinct from E. coli, where recombinant proteins tend to accumulatewithin inclusion bodies, and then require solubilization and refolding.

Amino terminal sequencing was done by standard Edman degradation, andconfirmed the N-terminal sequences discussed above.

4D. Bioactivity of hGH Expressed in Plant Plastids

Bioactivity of the pH adjusted and centrifuged extract was tested usingcells from an Nb2 cell line. These cells proliferate in the presence ofgrowth hormone and other estrogenic type compounds. The assay involvesputting various concentrations of growth hormone-containing extract intoa 96 well plate. Then a constant amount of cells are added to each well.The plate is incubated for 48 hrs and then a reagent called MTS isadded. Metabolizing cells take up the MTS and convert it to a bluecolored substance. The more cells there are the more blue color in thewell. The blue color is measured using a spectrophotometer. The numberof cells should be proportional to the concentration of growth hormonein the media. At some high concentration one expects that the cells willbecome saturated with growth hormone and that the dose response willlevel off. At very low hGH concentrations essentially no enhanced growthis seen. A sigmoidal shape graph is expected to be produced graphing thecell number (or absorbance) versus hGH concentration graph.

Proper disulfide pairing in the chloroplast hGH implies that the proteinshould be biologically active. To test this hypothesis in vitro, a ratlymphoma cell line, Nb2, that proliferates in the presence ofsomatotropin (hGH) and other estrogenic type compounds was employed.Proliferation of this cell line is proportional to the amount ofsomatotropin in the culture medium, until saturation is reached. The ionexchange column eluate from transplastomic Nt-4838 and Nt-38794 plantsor identically treated wild-type plants was added to the Nb2 cellculture medium. As control, E. coli produced, refolded hGH was used. Thewild-type plant extract showed no activity in this assay, indicatingthat there is no endogenous plant compound capable of stimulating growthof the Nb2 cell line. In contrast, the Nt-4838 and Nt-38794 extractsboth stimulated proliferation of the cell line to an equal extent as thepositive controls: either wild-type plant extract that had been spikedwith purified E. coli hGH or the pure hGH alone.

The Nb2 cell results show that the chloroplast derived hGH isbiologically active. Previous studies of recombinant somatotropinproduced in E. coli showed equivalent pharmacokinetics of the proteinwith either an N-terminal methionine or phenylalanine (Moore, et al.(1988) Endocrinology 122:2920-2926). In this study, ubiquitin cleavageof the fusion protein in Nt-38794 lines generated predominantly P-hST,suggesting that this species is also bioactive. The hST from Nt4838extracts was also characterized. Amino acid analysis indicated >95%protein species with alanine at the N-terminus. This result suggeststhat a methionine aminopeptidase activity generated the alanine-hST,which is also bioactive. A similar aminopeptidase activity exists in E.coli (Meinnel, et al. (1993) Biochimie 75:1061-1075). This finding inplastids may be exploited in the future as an alternative means togenerate a non-methionine N-terminus.

The results of the bioactivity assay (FIG. 13) demonstrates that the hGHexpressed from a plant plastid has a sigmoidal shape when graphed asabsorbance versus hGH concentration.

Example 5 Analysis of Aprotinin Transplastomic Tobacco Plants

5A. Western Analysis of Aprotinin Expression in Plastids

Homoplasmic tobacco lines expressing are used to determine theexpression of the aprotinin protein. Western blot analysis was performedon tobacco lines containing constructs pCGN6146, pCGN6147, pCGN6154 andpCGN6156 for plastid expression of aprotinin.

Total protein extractions and western blot procedures were performed asdescribed above, with the exception of the primary antibody was raisedagainst aprotinin.

The results of the Western analysis is shown in FIG. 14. These resultsindicate that aprotinin is expressed from the T7 polymerase promoterwhen the aprotinin coding sequence is fused with either the PetA or fulllength GUS gene. Furthermore, these results indicate that the petAsequence efficiently targets the aprotinin protein to the plant cellthylakoid.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claim.

1. A method for producing a non-methionine N-terminus protein in aplastid, wherein said method comprises: transforming a plastid with aconstruct comprising, as operably joined components in the 5′ to 3′direction of transcription, (a) a promoter functional in a plastid, (b)a DNA sequence encoding a cleavable ubiquitin peptide, (c) a DNAsequence encoding a protein of interest, (d) a transcription terminationregion, and (e) at least two DNA regions of homology to the genome ofsaid plastid flanking said operably joined components of (a), (b), (c)and (d); and growing a plant cell comprising said transformed plastidunder suitable conditions for expression of said protein of interest andsaid cleavable ubiquitin sequence in said plastid.
 2. The methodaccording to claim 1, wherein said construct further comprises (f) agene encoding a selectable marker for selection of a plant cellcomprising a plastid expressing said marker.
 3. The method according toclaim 1, wherein said construct further comprises (g) a ribosome bindingsite joined to said promoter (a).
 4. The method according to claim 3,wherein said ribosome binding site is derived from a leader sequenceselected from the group consisting of a plastid leader sequence, abacterial leader sequence and a bacteriophage leader sequence.
 5. Themethod according to claim 3, wherein said ribosome binding site isselected from the group consisting of the binding site of the T7bacteriophage gene 10 leader and the rbcL RBS.
 6. A plastid having aprotein of interest produced according to the method of claim
 1. 7. Theplastid according to claim 6 wherein said construct further comprises(g) a T7 bacteriophage gene 10 leader ribosome binding site that isoperably joined to a plastid promoter, and wherein said protein ofinterest comprises at least about 1.0% of total soluble protein in saidplastid.
 8. The plastid according to claim 7 wherein said protein ofinterest comprises at least about 7.0% of total soluble protein in saidplastid.
 9. A plastid comprising stably incorporated and operably joinedcomponents (a), (b), (c), and (d) contained within said DNA regions ofhomology of the construct of claim 1 within its genome.
 10. A plant cellcomprising a plastid comprising stably incorporated and operably joinedcomponents (a), (b), (c), and (d) contained within said DNA regions ofhomology of the construct of claim 1 within its genome.
 11. A plant cellcomprising a transformed plastid produced according to the method ofclaim
 1. 12. A plant, plant seed or plant part each comprising a plastidaccording to claim
 6. 13. A plant, plant seed or plant part eachcomprising a plastid according to claim
 9. 14. A plant, plant seed orplant part each comprising a plant cell according to claim
 10. 15. Aplant, plant seed or plant part each comprising a plant cell accordingto claim
 11. 16. The method according to claim 1 wherein said protein ofinterest is folded with the correct number of disulfide bonds.
 17. Themethod according to claim 1 wherein said protein of interest is hGH. 18.The method according to claim 1 wherein said protein of interest isbioactive when isolated from said plant cell.
 19. The method accordingto claim 18 wherein said protein of interest is hGH.
 20. A plastidcomprising: operably joined components (a), (b), (c), and (d) containedwithin said DNA regions of homology of a construct according to claim 1stably integrated into its genome; and a non-methionine N-terminusprotein.
 21. A plant cell comprising a plastid having: operably joinedcomponents (a), (b), (c), and (d) contained within said DNA regions ofhomology of a construct according to claim 1 stably integrated into itsgenome; and a non-methionine N-terminus protein.